Gene Check has developed a method of SNP detection, RecA Mediated Ligation (RML), that combines the extremely precise homology searching ability of the E. coli recombination protein, RecA, with the proven SNP detection method, oligonucleotide ligation. RML is an extremely powerful, yet simple and low cost method of SNP detection. It is the goal of this project to adapt the RML method to a microarray format and to combine it with Rolling Circle Amplification (RCA) to allow direct detection of SNPs from genomic DNA. RML products will be immobilized by hybridization to sequence specific oligonucleotides on individual microarray spots. The immobilization oligonucleotides will be designed with short regions of complementarity to each of the RML oligonucleotides to allow immobilization only of RML products and not of the component RML oligonucleotides. Following hybridization to an array, RML products will be detected by means of allele specific RCA. Allele specific RML oligonucleotides will be prepared with "universal" RCA primer sequences additions. Two RCA circles, with sequences complementary to these primer sequences will be used to extend the RML products and to create sequences for decorator probe annealing. By using different decorator probe sequences on the two RCA circles, it will be possible to genotype any number of two-allele SNPs on a single array. Four well-characterized SNPs from sheep genomic DNA will be genotyped individually and multiplexed. The RML-RCA method will be the most powerful method of SNP genotyping currently available and will have immediate and wide spread application. It will allow direct genotyping from small amounts of genomic DNA (on the order of 100ng), will not require denaturation or amplification of target DNA, will be capable of extremely high order multiplexing (greater than 1000 SNPs from a single sample in a single tube), and will be able to genotype large numbers of SNPs rapidly and inexpensively. [unreadable] [unreadable] [unreadable]